Biology subjects

Bramkamp, M.

Publications and source records attributed to Bramkamp, M..

24 records · Page 2Linked to original sources

bioRxiv2020

Lsr2 is a nucleoid-associated protein that exerts pleiotropic effects on mycobacterial cellular processes

Lsr2 is involved in maintaining chromosome structure in asymmetrically dividing mycobacteria and is essential in the tubercle bacillus (M. tuberculosis) during infection. Here, we report that a lack of Lsr2 profoundly impacts the mycobacterial cell morphology and the properties of the cell envelope resulting in the formation of smooth, short and antibiotics sensitive cells. Lsr2 forms large and dynamic nucleoprotein complexes in vivo and deletion of lsr2 gene exerts a profound effect on the replication time and replisome dynamics. We suggest that the Lsr2 nucleoprotein complexes may contribute to maintaining the proper organization of the newly synthesized DNA. Moreover, we demonstrate that the N-terminal oligomerization domain of Lsr2 is indispensable for the formation of nucleoprotein complexes in vivo. Collectively, our results indicate that Lsr2 exerts a pleiotropic effect on cellular processes and appears to be an attractive target for the development of a novel antitubercular drugs.

microbiology↗

bioRxiv2019

Membrane fluidity controls peptidoglycan synthesis and MreB movement.

The bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins. We show here that bacterial flotillins are important factors for membrane fluidity homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In vitro, the addition of a flotillin increases membrane fluidity of liposomes. Our data support a model in which flotillins are required for direct control of membrane fluidity rather than for the formation of protein complexes via direct protein-protein interactions.

microbiology↗

bioRxiv2019

Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

Higher-order chromosome folding and segregation is tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remains poorly characterized, especially in non-model species. Here, we investigate the role of DNA partitioning protein ParB and condensin complexes, two key players in bacterial chromosome structuring, in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for a reliable chromosome segregation. Using a combination of chromatin immunoprecipitation and photoactivated single molecule localization microscopy evidences the formation of distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further identified and functionally characterized two condensin paralogs. Whereas SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while showing that some condensin complexes evolved functions uncoupled from chromosome folding.

microbiology↗

bioRxiv2018

Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

The dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

biochemistry↗

bioRxiv2018

Substrate-dependent cluster density dynamics in bacterial phosphotransferase system permeases

Bacteria take up carbohydrates by membrane-integral sugar specific phosphoenolpyruvate-dependent carbohydrate:phosphotransferase systems (PTS). Although PTS is at the heart of bacterial carbon uptake and centrally involved in regulation of carbon metabolism, little is known about localization and putative oligomerization of the permease subunits (EII) of PTS. Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters from C. glutamicum using widefield and single molecule localization microscopy. PtsG and PtsF form membrane embedded clusters that localize in a punctate pattern within the cell membrane. The size, number and fluorescence of the observed clusters changes upon presence or absence of the transported substrate. In presence of the transport substrate clusters significantly increased in size. Photo-activated localization microscopy (PALM) data revealed that, in presence of different carbon sources, the number of EII protein events per cluster remain the same, however the density of PTS molecules within a cluster reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transport substrate the EII proteins in individual clusters occupy larger membrane areas, thereby decreasing protein density in individual clusters. This mechanism allows for efficient use of the limited membrane space under varying growth conditions without need of protein removal and re-synthesis.\n\nImportanceThe carbohydrate transport system PTS is centrally involved in the regulation of sugar metabolism. Although much is known about the regulatory interaction, the genetic control and the structure/function relationship of the individual PTS components, we know almost nothing about the spatio-temporal organization of the PTS proteins within the cell. We find dynamic clustering of PTS permeases in Corynebacterium glutamicum. Using single molecule resolution photo-activated localization microscopy we could show that PTS EII protein cluster are dynamically changing protein density upon substrate availability. Our findings imply a novel strategy of regulating limited membrane space efficiently. Furthermore, these data will provide important insights in modelling carbohydrate fluxes in cells, since current models assume a homogeneous distribution of PTS permeases within the membrane.

microbiology↗

bioRxiv2016

Novel chromosome organization pattern in actinomycetales-overlapping replication cycles combined with diploidy

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we find that C. glutamicum cells are at least diploid under all conditions tested and that these organisms have overlapping C-periods during replication with both origins initiating replication simultaneously. Based on experimentally obtained data we propose growth rate dependent cell cycle models for C. glutamicum.

microbiology↗